Preparing Method for Xanthophyll Crystals with Higher Content of Zeaxanthin from Plant Oleoresin

ABSTRACT

The invention makes public a preparing method for xanthophyll crystals with higher content of zeaxanthin from plant oleoresin. The current methods generally are to get quite pure crystal forms of xanthophyll or zeaxanthin, and they refer to several separation steps. The invention mixes the xanthophyll diester-containing plant oleoresins and food grade alcohol solvents to form even solution, and then soap-dissolve the solution under an alkaline environment; then replenish organic solvents and emulsifiers into the reaction solution and drop some alkali solution into the solution to make partial xanthophyll crystals be transformed to be zeaxanthin through epimerization reaction; after the reaction is finished, add the mixed solvents of alcohol solvent and water to separate out the crystals; use the method of centrifugation or filtration to get the crystals; wash the crystals several times with the mixed solution of deionized water and alcohols to remove the impurities among the crystals; recrystallize the gained crystals with absolute ethyl alcohol, and then dry the crystals to get the products. The invention can gain mixture crystals that contain xanthophyll and zeaxanthin at one time in quite high collection rate, and it is convenient for the followed product application.

FIELD OF THE INVENTION

The invention refers to the preparation of carotinoid, and in detail, itis a preparing method for xanthophyll crystals with higher content ofzeaxanthin from plant oleoresin (especially, marigold oleoresin).

BACKGROUND OF THE INVENTION

In nature, the carotinoid is a kind of widely existed material with acolor from yellow to red. In different fruits and vegetables, thereexists different kinds of carotinoid, for example, there existsβ-carotenes in carrots, there exists xanthophyll in marigold flowers,there exists zeaxanthin in straw berries, there exists lycopenes intomatoes, and chili class plants are quite rich in capsanthin andcapsochrome. Some dark color fruits and vegetables, the yolk, some fishclass carapaces, the bird class, the seaweed and the bacterium are quiterich in carotinoid, and the xanthophyll class content is also quiterich.

In recent years, some animal and human trials have proved the beneficialfunction of carotinoid. In general, carotinoid can be divided into twosubclasses, and they are the xanthophyll class or oxic class carotenewith relatively quite strong polarity, such as xanthophyll, zeaxanthin,astaxanthin, and so on, as well as nonpolar carbon and hydrogen classcarotene, such as β-carotene, lycopene, and so on. It at least containsnine conjugated double bonds among the carotinoid of the twosub-classes, these conjugated double bonds not only endow the carotinoidcolor feature, but also make it own quite strong antioxidation functionfor the prevention and cure of disease, and they can prevent cancer,arteriosclerosis, cataract, spot degradation, as well as other categoryof diseases. Due to the capability of carotinoid to eliminate efficientreactive oxygen free radicals and to prevent the generation of freeradicals, they can limit the oxidizing destruction effect of freeradicals.

For all carotinoid, due to their potential capability to prevent onekind of age related macular degenerations (ARMD), scientists and thepublic have paid more and more attention to the xanthophyll andzeaxanthin. Xanthophyll and zeaxanthin are the only carotinoid existingwithin the spot area (macular degeneration) of human being's retina, andthe area is closely related to the visual sensitivity of human beings(Bone et al. Invest. Ophthamal. Vis. Sci. 34:2033-2040, 1993). It canreduce the risk of getting aged macular degeneration by 43% to usuallyeat the fruits and vegetables that are rich in xanthophyll andzeaxanthin (Seddon et al. J. Am. Med. Assoc. 272:1413-1420, 1994), andthe metabolic pathway of these compounds for preventing aged maculardegeneration has been clear at present. The Food and Drug Administrationalso considers xanthophyll and zeaxanthin as “generally recognized assafe” (GARS). Therefore, these carotinoid can be used separately ortogether with other materials as nutritional supplements and foodcoloring agents, as well as be used clinically for preventing agedmacular degeneration and cancer, etc. The constitutional formulas ofxanthophyll and zeaxanthin are as follows respectively, they areisomers, the only difference between zeaxanthin and xanthophyll is theposition of double bonds on one (not two) end ring, the position of thedouble bonds on the two end rings of the former is symmetrical, but thatof the latter is asymmetrical, i.e. the entire straight chain part ofthe xanthophyll and zeaxanthin' each molecule is the conjugationstructure that owns alternant double bonds and single bond. In themolecule of zeaxanthin, the conjugation constitution extends to thefirst bond on the two end rings, but the conjugation extent ofxanthophyll is lower, because its double bonds on one of its end ringsdo not form the correct arrangement of complete conjugationconstitution, and it just is the difference of molecular structure thatresults in a certain difference between xanthophyll and zeaxanthin infunction and feature.

As a kind of natural pigments, xanthophyll and zeaxanthin exist widelyin nature, they mainly exist in higher plants, algae, fishes, shellclass and bacterias, and they exist in the forms of ester in the body ofliving beings. Among theses living beings, the marigold flower is a kindof good source for xanthophyll and zeaxanthin, there usually are about 2g xanthophyll class materials in 100 g marigold fresh flowers, the mainmaterials are xanthophyll that is more than 90%, and the rest arezeaxanthin and a few other carotinoid. It is the same as marigoldflowers, in other kinds of higher plant and algae sources, theproportion of xanthophyll is more compared with zeaxanthin, but thevolume of zeaxanthin in corns is more than that of xanthophyll. Inaddition, from the molecular structure it can find that there isalloisomerism between the xanthophyll and the zeaxanthin, the sourcesare different, and the stereoisomerism constituent is also different,for example, they mainly are (3R,3′R,6′R)-xanthophyll or(3R,3′R)-zeaxanthin in plant resources, but xanthophyll exist in formsof (3R,3′R,6′R)—, (3R,3′R,6′S)—, and (3R,3′S,6′S)— in animal resources,such as in fishes and shells.

Since it refers to several steps of reactions to get xanthophyll throughchemical synthesis, it may cost a lot of time and strength, and the costis very high. The quite economic way that is easy for mass production toget plenty of xanthophyll crystals is to get them from natural resourcesthrough extraction, separation and purification.

Many vegetables and fruits, such as spinaches, broccolis, cabbages andcorns, etc, are quite rich in xanthophyll, but marigold flowers andcalendulas are the richest sources of xanthophyll, of course, there alsoexists other carotinoid in these plants. The xanthophyll in plantsusually exists in forms of single ester and double ester which usuallyis formed through esterification between the xanthophyll and someC₁₂-C₁₈ long-chain aliphatic acids.

Generally, we use organic solvents to extract xanthophyll ester fromplants, preferentially from marigold flowers, calendulas and other darkgreen vegetables, and these organic solvents are quite easy to beseparated. The marigold peaberry extract (marigold oleoresin) is a verygood xanthophyll ester source, and the other classes of carotinoid arerelatively not rich in it. After it is hydrolyzed under alkalineconditions, the xanthophyll crystals will be dissociated, and thexanthophyll crystals will be further purified after the fatty acid saltgot from the soap-dissolving process is washed off

Meanwhile, as the above description, since there mostly exists aconjugated double bond in the molecular structure, which makes thezeaxanthin have stronger antioxidant activity than the xanthophyll, andthe zeaxanthin takes a more important effect for the health of humaneyes. In fact, some researches in the middle to late 1980s had alsoproved that they mainly were zeaxanthin in the small area at the centerof human eyes' macula lutea. They leave the concave concentrically andget close to the circum of the macula lutea, so the quantity ofzeaxanthin gradually becomes less, and the quantiy of xanthophyllgradually becomes more. At the circum of the macula lutea, xanthophyllis the main xantheins. This also can be found from the proportion changebetween xanthophyll and zeaxanthin in different parts of natural andhuman tissues, in the marigold flowers, the raw materials ofxanthophyll, the proportion between xanthophyll and zeaxanthin is about10-12:1, the proportion is about 3-5:1 in human blood, the proportion is3:1 at the circum of retina macula lutea, but at the center of maculalutea the proportion is completely opposite, and it is 1:3. The recentresearches have found that in an isomer of zeaxanthin at the centralarea if macula lutea, the internal compensation (3R, 3′S,meso)—zeaxanthin took quite a lot proportion, and the proportion will beless, if it is closer to the periphery. More and more evidences haveproved that the internal compensation zeaxanthin in macula lutea are gotthrough epimerization transposition, because it almost can not detectthis kind of isomers of zeaxanthin in nature, human blood and otherhuman tissues.

The distribution proportion change of xanthophyll and zeaxanthin inhuman eyes just proved the important and unique effects of zeaxanthin,in particular, the internal compensation zeaxanthin, for the health ofhuman eyes. Actually, many human experiments also have proved that itwill take a better effect for the complex use of xanthophyll andzeaxanthin, so most the eye care products that contain xanthophyll onthe market usually are added with certain quantity of extra zeaxanthin,i.e. it adds with xanthophyll and zeaxanthin respectively according tocertain proportion in the application partition. However, the respectiveaddition of xanthophyll and zeaxanthin must bring many unnecessarytroubles for the purchase of raw materials, the operation of productionprocess and product quality control. If it can make some part of thexanthophyll form zeaxanthin through epimerization during the process ofmaking xanthophyll, it can ensure that it contains xanthophyll andzeaxanthin at the same time in the products and keeps the requiredproportion. Thus, it can just add one kind of carotinoid agents thatcontains certain proportion of xanthophyll and zeaxanthin at the sametime, and this will result in much convenience during the subsequentproduct application process.

At present, there also are some patents and essays that refer to themethod to separate xanthophyll crystals in large scale from the peaberryof marigold or the method to gain zeaxanthin crystals through theepimerization transposition of xanthophyll. The targets of these essaysgenerally are to get quite pure crystal forms of xanthophyll orzeaxanthin, and they refer to several separation steps.

In U.S. Pat. No. 5,382,714, it separates and purifies xanthophyllthrough washing the soap-dissolved marigold oleoresin under a quite lowtemperature and crystallizing the mixed solvent under a low temperature.The purification process not only is time-consuming, but also useschlorinated organic solvents, so the products gained are not fit forbeing used in foods and medicines. The proportion between xanthophylland zeaxanthin in the products do not change much compared with the rawmaterials.

In U.S. Pat. No. 5,648,564, it makes public a method to separatexanthophyll crystals, it soap-dissolves xanthophyll diester-containingthe propylene glycol solution of marigold oleoresins at first, and thenit will recrystallize them. There are also several defects in thisprocess: the first, since the viscosity of propylene glycol is quitestrong, it requires a quite high temperature during the soap-dissolvingprocess or the followed process, the entire system needs to be keptabove 70° C. for about 10 hours, this obviously is disadvantageous forthe stability of xanthophyll, the cis-trans isomers of xanthophyll alsowill change, what's more, the followed separation processes, such ascentrifugation and filtration, are also quite difficult; the second, thecollection rate of xanthophyll is quite low, it only is about 59%, andthe xanthophyll content in the products is also not high.

In U.S. Pat. No. 6,262,284, it describes a method to use tetrahydrofuranto extract and soap-dissolve carotinoid from marigold dry flowers, ituses plenty of organic solvents during the process, these solvents areharmful to the stability of xanthophyll, and it may result indeterioration that is caused by overoxidation.

In U.S. Pat. No. 6,329,557, it describes a method to extract xanthophyllcrystals from marigold oleoresins through large industrial scale. Thedefect of the process is to use plenty of organic solvents, such asnormal hexanes, ketones, etc, and these solvents are not fit for usingin foods.

In U.S. Pat. No. 6,380,442, it reports a method to separate carotinoidfrom plants, the method is also not attractive for industrialproduction, because it will use a lot of water (at least 30 times theraw materials) during the production process, and the operation is quitedifficult.

In U.S. Pat. No. 6,743,953, it uses organic solvents to separate andpurify xanthophyll from marigold oleoresins. It uses several organicsolvents, such as ipa, ethyl acetate, normal hexane, acetone andmethanol, etc, during the process, the operation is miscellaneous, theorganic solvent consumption is quite large, and the collection rate islow. Therefore, this method is also not fit for industrial production.

In U.S. Pat. No. 7,271,298, it makes public a method that uses absoluteethyl alcohol as the solvent to get higher content xanthophyll crystalsin high collection rate through simple technology, and this method alsodo not consider to enhance the proportion of zeaxanthin for the gainedcrystals.

In U.S. Pat. No. 5,780,693(CN1,178,787A), it designs a routine toproduce zeaxanthin taking xanthophyll as the raw materials. In general,it mainly uses dimethyl sulfoxide or the mixture of dimethyl sulfoxideand saturated alkane and/or arene organic solvents as the solvents anduses alkali hydroxides as catalysts to produce zeaxanthin throughtransposition of xanthophyll. Furthermore, it uses the organic solvents,such as normal hexanes, normal heptanes, dichloromethane, methylalcohols, etc, during the reaction process. It is obviously improper touse these toxic solvents to produce food grade or medicine gradezeaxanthin.

In U.S. Pat. No. 7,485,738, it uses xanthophyll as the raw materials togain high purity internal compensation zeaxanthin through epimerizationwith the catalysis of strong organic alkali, and the gained zeaxanthincrystals through this technology do not contain or contain very fewxanthophyll.

In a word, the described methods in the above patents have severaldefects as follows: 1) it uses some toxic organic solvents during theprocesses, it is quite difficult or impossible to remove these solventscompletely, and this results in that the produced xanthophyll orzeaxanthin crystals are unfit for being used as edible products of humanbeings; 2) or the used organic solvents are quite high in viscosity, theoperation during the process of separation and purification is quitedifficult, in order to gain higher content crystals, it needs atreatment process with several steps, so it is not suitable forindustrial production; 3) or the collection rate of carotinoid is quitelow, since it refers to several steps during the process, it results inthe low product collection rate, and it is only about 50%. Inparticular, when it uses marigold flowers as the raw materials to gainthe mixtures of zeaxanthin and xanthophyll through isomerizationreaction to transform xanthophyll crystals partially after it gainedxanthophyll crystals through soap-dissolving reaction, separation andretification, the collection rate is lower. 4) or the singleconcentration of xanthophyll or zeaxanthin in the products is quitehigh, they can not reach the purpose to adjust xanthophyll andzeaxanthin concentration through controlling the reaction processaccording to the requirements. Therefore, it is necessary to find amethod that is suitable to produce high purity xanthophyll andzeaxanthin in industrial scale, which uses as few as possible toxicorganic solvents, refers to as few as possible steps, owns quite highcollection rate, and can control the reaction parameters according tothe needs, so that it can reach the purpose to adjust the xanthophylland zeaxanthin concentration in the products.

SUMMARY OF THE INVENTION

In order to overcome the defects of the above described methods, theinvention provides a preparing method for xanthophyll crystals withhigher content of zeaxanthin from plant oleoresin, and it can achievethe purpose to be convenient and to adjust the zeaxanthin content inxanthophyll crystals effectively.

Therefore, the invention adopts the following steps:

a) mix the xanthophyll diester-containing plant oleoresin and food gradealcohol solvents through stirring, and then it will form uniformfree-pouring mixed liquor. Next, soap-dissolve this mixed liquor 3-5hours under an alkaline environment, i.e. with a temperature between 40°C. and 85° C.

b) replenish the mixed liquor got from step a) with organic solvents andemulsifier and stir it to be even;

c) drop strong alkaline solution into the mixed liquor got from the stepb) to result in the epimerization of partial gained xanthophyll crystalsto transform them to be Zeaxanthin, and then adjust the reaction timeand temperature in accordance with the required proportion between thexanthophyll and zeaxanthin;

d) use the mixed solvents consisting of deionized water and alcoholssolvents to dilute the reaction solution got from step c), which canmake the volume concentration of the solute in the solution be loweredto 10-50%, the dosage of deionized water and alcohols solvents are 2-10times and 0.5-2 times the weight of raw material plant oleoresinrespectively, and then heat up to 60-75° C. and stir 0.5-2.0 hoursslowly to make carotinoid be separated out in form of granular crystals;

e) use the method of centrifugation or filtration to get the crystalsformed in step d);

f) use 60-85° C. hot water to rinse the crystals got from step c) 2-3times until the mother liquor is close to be colorless;

g) In order to make the final product be easy to be dried, for thedrying before desiccation, use absolute ethyl alcohol to drip-wash thefilter cake one time, and then use vacuum drying or freeze drying to getxanthophyll crystals, which can make its final loss on drying be lessthan 5%.

The used plant oleoresins in the invention are extracts of marigoldflowers, calendulas, spinachs, strawberries, broccolis, corns, cabbages,in these raw materials, the xanthophyll content in marigold flowerextracts is relatively quite high, and they are the preferential rawmaterials. Due to the difference of varieties, planting conditions,harvest periods and extract methods, these oleoresins contain 5-30%xanthophyll diesters and a few other carotinoid, such as all-transzeaxanthin, α- and β-cryptoxanthin and β-carotenes, etc.

According to ultraviolet visible ray spectropho-tometry analysis andHPLC analysis, we know that the final products contain 75-95%carotinoid, among of them, there are 15%-95% all-trans xanthophyll,85%-5% zeaxanthin, 0.1-1.0% other possible geometric isomers, as well asless than 1.0% other carotinoid. There is completely no need to worryabout the harm of this microscale other carotinoid, because they arealso food sources, and their concentration in human blood serum is muchhigher compared with xanthophyll.

In the produced final crystallization products through this technology,there are no residuals of toxic organic solvents and other toxiccompounds, so it is suitable to be used as foods of human.

In step a) of the invention, it usually dissolves the plant oleoresinsin the food grade alcohol solvents that is 0.5-2.0 times that of theplant oleoresins in volume, and through stirring, xanthophyll esters andother kinds of impurities, such as waxes, resins, other carotinoid andpigments, etc, are dissolved or dispersed in these solvents to form akind of even solution; Add aqueous alkali solutions (such as NaOH, KOH,sodium methoxide or sodium ethoxide) into the even solution to carry outsoap-dissolving, the needed quantity of alkali is 0.5-6.0 times theweight of xanthophyll diester-containing oleoresins, xanthophyll,zeaxanthin and other carotinoid are dissociated out of the solutionduring this process, at the same time, the fatty acid in plantoleoresins (such as myristic acid, palmitic acid, stearic acid, etc)react with sodium or potassium to form soda soap or potash soap, theused food grade alcohol solvents are carbinols, ethyl alcohols,isopropyl alcohols, propyl alcohol, etc, and ethyl alcohols are apreferential option.

In the step b) and c) of the invention, it replenishes a certainquantity of organic solvents to enhance the solubility of thedissociated carotinoid in this solution, the dosage of organic solventsare 1.0-3.0 times the volume of the same plant oleoresin weight, theorganic solvents can be alcohols solvents, such as ethanol, isopropylalcohol, propanol, propylene glycol, etc, they also can be esterssolvents, such as ethyl acetate, ethyl ester isobutyl paraben, etc, andthey also can be other organic solvents, such as dimethyl sulfoxide,methylene chloride, etc. It can make carotinoid be dissolved better toadd a certain quantity of emulsifiers, the used emulsifiers can be Tweenemulsifiers, such as Tween-60, etc, they also can be Span emulsifiers,such as Span-40, etc, the dosage of emulsifiers is 0.4-1.0 times theweight of xanthophyll crystals in plant oleoresins, in this way, afterit is replenished certain quantity of strong alkalis (the dosage ofstrong alkalis is 0.1-1.0 times the weight of xanthophylldiester-containing plant oleoresins) through dropping, it does not needextra steps to separate out the xanthophyll crystals, and it candirectly make partial xanthophyll be transformed to zeaxanthin throughepimerization. The proportion between the xanthophyll and zeaxanthin inthe reaction products can be monitored through using high performanceliquid chromatography on the samples. In accordance with the requiredproportion between the xanthophyll and the zeaxanthin, it can adjust thereaction time and temperature, the selection scope of reaction time is0.5-6.0 hours, and the selection scope of temperature is 60-90° C.

In step e) of this invention, it separates out the formed crystals instep d) through traditional separation process, such as centrifugation,filtration and pressure filtration, etc, before separating the crystals,in order to further reduce the concentration of crystals to make thefollowed separation operation process be easy to be carried out, it canuse proper quantity of hot water to dilute the solution. After theseparation operation, the impurities, such as fatty acids, salts, soaps,water-solubility chlorophylls and flavones, are transferred into themother liquor, and the crystals, such as xanthophyll and zeaxanthin, areleft in the filter cake. What need to be explained is that the purity ofthe final products, the proportion between xanthophyll and zeaxanthin,the collection rate of carotinoid crystals not only depends on theaddition of the solvents during the soap-dissolving process and theaddition of the alcohol solvents during the dilution process, as well asthe addition, reaction time and reaction temperature of alkalines duringthe isomer reaction process, but also depends on the crystallizationtime before washing.

If the proportion of solvents is too low during the soap-dissolvingprocess, it will make the viscosity of the solvents be quite strong, andthis is not good for the followed separation operation; if theproportion of the solvents is too high, it will make the concentrationof xanthophyll in the solution become low, and this is not good forcomplete soap-dissolving, which will lower the collection rate of theproducts and the content of crystals. This is why partial organicsolvents need to be replenished before isomerization reaction, becausethe solubility of oleoresins is relatively quite large, and what gainedafter soap-dissolving reaction are carotinoid crystals, the solubilityof these crystals in organic solvents will be lowered a lot, but it mustmake them be dissolved and take part in reaction in order to make it betransformed to be zeaxanthin, so it must replenish certain quantity oforganic solvents after the soap-dissolving reaction is finished andbefore the isomerization reaction is started. On the one hand, it willnot dilute the concentration of oleoresins before soap-dissolvingreaction, and it can make the carotinoid oleoresins finishsoap-dissolving within quite short period to enhance the carotinoidcontent in the final products, on the other hand, it can also ensure thesufficient dissolution of the reactants, i.e. the dissociativecarotinoid crystals, so that it can make the isomerization reaction becontrollable.

It is also very important to add in alkalis bath by bath at differentreaction stages. Before the soap-dissolved plant oleoresins getdissociative carotinoid crystals, but these crystals does not bedissolved in the organic solvents, so that it can carry outisomerization reaction, the concentration of alkalis can not be toohigh, otherwise partial carotinoid oleoresins and generated carotinoidcrystals will be lost through the oxidization of high concentrationalkali, and it even will be carbonized for a serious situation. It canavoid this situation to add different quantity of alkalis in differentstages. The purpose to add strong alkalis through dropping during thelater process of transforming xanthophyll into zeaxanthin throughisomerization is also because of this.

Before the isomerization reaction, it is also good for the occurrence ofisomerization reaction to add certain quantities of emulsifiers. Sinceit needs to add certain quantities of aqueous alkalis as the catalyst ofthe reaction during the process of soap-dissolving and isomerizationreaction, inevitably, it will bring in a little water, and the watercertainly will lower the solubility of the carotinoid crystals inorganic solvents greatly. As a result, it lowers the rate ofisomerization reactions. It can reduce the influence of this water tothe carotinoid crystals to the least extent through adding certainquantities of emulsifiers, and it can enhance the solubility ofcarotinoid, so that it will be good for the occurrence of the reaction.Furthermore, a few of emulsifiers can increase the contact opportunitybetween the catalysts and carotinoid crystals, and it will make theisomerization reaction be easier to be carried out.

In addition, the rectification process of alcohol solution beforeseparating the final carotinoid crystals will also influence the contentand collection rate of the products. It will lower the collection rateof the products to add in too many alcohols during the dilution process,and it will increase the operation difficulty to add too less alcohols.The time to precipitate crystals also can not be too short, otherwise,it will lower the collection rate.

The advantages of the invention are as follows: 1) it uses plantoleoresins as the raw materials, it can get the crystals that containxanthophyll and zeaxanthin at one time through two continuous reactions,i.e. the soap-dissolving reaction and isomerization reaction, theproportion between xanthophyll and zeaxanthin in the crystals can beadjusted through controlling reaction conditions in accordance with therequirements, it is convenient for the followed product application, andit can effectively avoid the inconvenience in purchasing, storage andoperation, because it needs to mix the xanthophyll and the zeaxanthinrespectively while applying the products. 2) It can gain mixturecrystals of xanthophyll and zeaxanthin with quite high collection ratethrough a series of condition optimizing, it does not need extraoperation to purify the xanthophyll crystals during the reactionprocess, and it can carry out isomerization reaction directly, and thisis much higher in collection rate compared with the current technologythrough which it will get higher purity xanthophyll crystals at first,and then it can get the mixture crystals of xanthophyll and zeaxanthinthrough the isomerization of partial xanthophyll crystals; 3) Adding theorganic solvents and base catalysts batch by batch, it not only canprotect the normal occurrence of the reaction, but also can shorten thereaction time as much as possible and ensure the complete reaction, andit can lighten the destruction effect of the strong alkalis to thereaction products and substrates; 4) During the occurrence stage of theisomerization reactions, it can enhance the solubility of carotinoidcrystals in the organic solvents, and it can effectively increase thecontact opportunities between the base catalysts and the reactionsubstrates to make the reaction be more complete. 5) Since the usedsolvents during the process are water and low grade alcohols or esters,their viscosity is quite low, the next separation operation process isrelatively quite easy, and it does not use extra organic solventrecrystallization rectification process. In a word, the described methodin the invention is very economic and is suitable for large scaleindustrial production.

Next, it is the detailed introduction to the invention according to theconcrete implementation methods.

DETAIL DESCRIPTION OF THE INVENTION

Implementation Example 1

Mix 1000 g marigold oleoresins (total xanthophyll content is 15.2%) and2000 mL isopropyl alcohol, heat them to 40° C., stir them until theybecome a kind of even flowing solvent, drop 405 mL 45% NaOH solutioninto them slowly while stirring them, the dropping time is 60 minutes,and let it carry out soap-dissolving 5 hours under this temperature.

Heat the reaction solution to 60° C., add isopropyl alcohol solution2400 mL, emulsifier Tween-80 80 g, and then stir it 0.5 hour to make itbe mixed evenly. Drop 37% CH₃ONa solution 240 mL slowly, the droppingtime is 1.0 hour, heat it to 70° C. at the same time, and stir it tomake it react for 0.5 hour.

Add 2000 mL deionized water and 200 mL ethyl alcohol after the reactionis finished, keep the temperature of the solution at 60° C. during thedilution process, and stir it slowly for 30 minutes. Separate the gainedcrystals through centrifugation, the process is quite easy, and it canbe finished within 10 minutes. Wash the collected crystals 2-3 timeswith 60° C. hot water until the eluate is almost colourless, wash thecrystals with dropping absolute ethyl alcohol at last, and then dry itin vacuum under 40° C. until its loss on drying is less than 5%.

At last, we can get 144.6g finished products, 87.2% of them are totalcarotinoid (it can be analyzed through ultraviolet visible raysluminosity instruments), it contains 95.1% all-trans xanthophyll inthese carotinoid, 4.8% all-trans zeaxanthin (it can be analyzed throughHPLC), and the rest are other microscale carotinoid. The collection rateof total carotinoid is 82.95%.

It does not contain toxic organic solvents in the products, and it issuitable to be used in the forms of nutritional supplements and foodadditives. The application form of this crystal can be oil suspension(it is emulsified through mixing with plant oil), peaberry (it is themicrocapsule gained through atomizing condensation), dry powder (it isthe microcapsule gained through atomizing drying), etc.

Implementation Example 2

Mix 500 g marigold oleoresins (total xanthophyll content is 14.5%) and250 mL propyl alcohol, heat them to 85° C., stir them until they becomea kind of even flowing solvent, drop 405 mL 45% NaOH solution into themslowly while stirring them, the dropping time is 60 minutes, and let itcarry out soap-dissolving 3 hours under this temperature. Cool thereaction solution to 60° C., add ethyl acetate solution 1500 mL,emulsifier Span-40 65 g, and then stir it 0.5 hour to make it be mixedevenly. Drop 50% sodium ethoxide solution 100 mL slowly, the droppingtime is 1.0 hour, heat it to 80° C. at the same time, and stir it tomake it react for 6.0 hour.

Add 1000 mL deionized water and 250 mL ethyl alcohol after the reactionis finished, keep the temperature of the solution at 60° C. during thedilution process, and stir it slowly for 120 minutes. Separate thegained crystals through filtration, the process is quite easy, and itcan be finished within 30 minutes. Wash the collected crystals 2-3 timeswith 85° C. hot water until the eluate is almost colourless, wash thecrystals with dropping absolute ethyl alcohol at last, and then dry itin vacuum under 40° C. until its loss on drying is less than 5%.

At last, we can get 61.0 g finished products, 85.6% of them are totalcarotinoid (it can be analyzed through ultraviolet visible raysluminosity instruments), it contains 15.2% all-trans xanthophyll inthese carotinoid, 84.8% all-trans zeaxanthin (it can be analyzed throughHPLC), and the rest are other microscale carotinoid. The collection rateof total carotinoid is 72.02%.

It does not contain toxic organic solvents in the products, and it issuitable to be used in the forms of nutritional supplements and foodadditives. The application form of this crystal can be oil suspension(it is emulsified through mixing with plant oil), peaberry (it is themicrocapsule gained through atomizing condensation), dry powder (it isthe microcapsule gained through atomizing drying), etc.

Implementation Example 3

Mix 1000 g marigold oleoresins (total xanthophyll content is 14.5%) and2500 mL ethyl alcohol, heat them to 75° C., stir them until they becomea kind of even flowing solvent, drop 380 mL 50% NaOH solution into themslowly while stirring them, the dropping time is 60 minutes, and let itcarry out soap-dissolving 4 hours under this temperature.

Heat the reaction solution to 60° C., add propyl alcohol solution 1200mL, emulsifier Tween-60 60 g, and then stir it 0.5 hour to make it bemixed evenly. Drop 37% sodium methoxide solution 100 mL slowly, thedropping time is 1.0 hour, heat it to 80° C. at the same time, and stirit to make it react for 3.0 hour.

Add 1000 mL deionized water and 250 mL ethyl alcohol after the reactionis finished, keep the temperature of the solution at 60° C. during thedilution process, and stir it slowly for 120 minutes. Separate thegained crystals through pressure filtration, the process is quite easy,and it can be finished within 30 minutes. Wash the collected crystals2-3 times with 85° C. hot water until the eluate is almost colourless,wash the crystals with dropping absolute ethyl alcohol at last, and thendry it in vacuum under 40° C. until its loss on drying is less than 5%.

At last, we can get 141.6 g finished products, 84.7% of them are totalcarotinoid (it can be analyzed through ultraviolet visible raysluminosity instruments), it contains 87.1% all-trans xanthophyll inthese carotinoid, 12.8% all-trans zeaxanthin (it can be analyzed throughHPLC), and the rest are other microscale carotinoid. The totalcollection rate of xanthophyll is 82.71%.

Implementation Example 4-7

Steps Implementation Implementation Implementation Implementation Itemsexample 4 example 5 example 6 example 7 The numbers of the raw materials300 g/14.5% 1500 g/13.6% 480 g/15.4% 600 g/14.6% (g) and the xanthophyllcontent among them The types and quantities of solvents methyl ethylnormal propyl ethyl while carrying out soap-dissolving alcohol/ alcohol/alcohol/ alcohol/ reaction 600 ml 1200 ml 500 ml 800 ml The types andquantities of base 37% sodium 50% sodium 50% KOH 45% NaOH catalystswhile carrying out methylate ethoxide ethyl alcohol solution/soap-dissolving reaction solution/ solution/ solution/ 400 ml 118 ml 816ml 1780 ml The types and quantities of ethyl ethyl isopropyl isobutylreplenishing solvents before the acetate/ alcohol/ alcohol/ acetate/isomerization reaction 300 ml 1500 ml 1200 ml 1800 ml The types andquantities of Tween-60/ Span-40/ Tween-80/ Span-20/ replenishingemulsifiers before the 17.5 g 204 g 52.0 g 88.0 g isomerization reactionThe types and quantities of 50% sodium 50% KOH 45% NaOH 37% sodiumdropping alkali liquor before the ethoxide ethyl alcohol solution/methylate isomerization reaction solution/ solution/ 330 ml solution/ 87ml 820 ml 50 ml The temperature of isomerization 90° C. 60° C. 70° C.85° C. reaction The time of isomerization reaction   3.0 hours   1.0hours   4.5 hours   6.0 hours The number of the final products (g) 42.9g  178.8 g   71.5 g  72.5 g  The total carotinoid content (%, 76.4%82.3% 74.8% 88.4% UV) in the final products The proportion between94.6%/5.2% 85.2%/14.5% 30.4%/69.4% 15.2%/84.7% xanthophyll andzeaxanthin in the final products (HPLC)

Compare the Implementation Examples

Soap-dissolve 1000 g marigold oleoresins (the total xanthophyll contentis 14.5%) in accordance with the technology conditions described in U.S.Pat. No. 7,271,298 and gain carotinoid crystals 129.6 g after removingthe related impurities through alcohol solution, the content ofcarotinoid crystals is 93.2%, the product collection rate is 83.3%, andthe carotinoid crystals contain 93.1% all-trans xanthophyll and 6.8%all-trans zeaxanthin.

Take the above described xanthophyll crystals 120 g to carry outepimerization reaction in accordance with the published method of U.S.Pat. No. 7,485,738, monitor the process of the reaction through HPLCchromatography, stop the reaction after 1.5 hours, remove the relatedimpurities through alcohol solution again, finally we can get carotinoidcrystals 71.3 g, the content is 85.6%, the proportion of all-transxanthophyll is 88.1%, the proportion of all-trans zeaxanthin is 11.8%,and the collection rate of epimerization reaction is 54.6%.

The total collection rate of the above two steps of reactions is only45.5%, and it is much lower than the product collection rate of theinvention.

1. A method for preparing xanthophyll crystals containing higher contentof zeaxanthin from plant oleoresin comprising following steps: a) mix axanthophyll diester-containing plant oleoresin and food grade alcoholsolvents through stirring, and then it will form uniform free-pouringmixed liquor. Next, soap-dissolve this mixed liquor 3-5 hours under analkaline environment, i.e. with a temperature between 40° C. and 85° C.b) replenish the mixed liquor got from step a) with organic solvents andemulsifier and stir it to be even; c) drop strong alkaline solution intothe mixed liquor got from the step b) to result in the epimerization ofpartial gained xanthophyll crystals to transform them to be Zeaxanthin;d) use the mixed solvents consisting of deionized water and alcoholssolvents to dilute the reaction solution got from step c), which canmake the volume concentration of the solute in the solution be loweredto 10-50%, the dosage of deionized water and alcohols solvents are 2-10times and 0.5-2 times the weight of raw material plant oleoresinrespectively, and then heat up to 60-75° C. and stir 0.5-2.0 hoursslowly to make carotinoid be separated out in form of granular crystals;e) use the method of centrifugation or filtration to get the crystalsformed in step d); f) use 60-85° C. hot water to rinse the crystals gotfrom step c) 2-3 times until the mother liquor is close to be colorless;g) for the drying before desiccation, use absolute ethyl alcohol todrip-wash the filter cake one time, and then use vacuum drying or freezedrying to get xanthophyll crystals, which can make its final loss ondrying be less than 5%.
 2. The method of claim 1, wherein it adopts theextracts of marigold flowers, calendulas, spinaches, strawberries,broccolis, cabbages or corns to make plant oleoresin.
 3. The method ofclaim 1, wherein the adding volume of food grade alcohol solvents is0.5-2.0 times the volume of the same weight of plant oleoresins in stepa).
 4. The method of claim 3, wherein the used strong alkalies are NaOH,KOH, sodium methoxide or sodium ethoxide, the needed volume of alkaliesis 0.5-6.0 times the xanthophyll diester-containing plant oleoresinswhile carrying out soap-dissolution, and the used volume of alkalies is0.1-1.0 times the weight of the xanthophyll diester-containing plantoleoresins when it has an isomerization reaction.
 5. The method of claim1, wherein it selects alcohol solvents or esters solvents for the addingorganic solvents, and the dosage is 1.0-3.0 times the volume of the sameweight of plant oleoresins.
 6. The method of claim 5, wherein it selectsethanol, isopropyl alcohol, propanol, ethyl acetate or ethyl esterisobutyl paraben for the adding organic solvents.
 7. The method of claim1, wherein it selects Tween emulsifier or Span emulsifier for thedescribed emulsifier, and its dosage is 0.4-1.0 times the weight ofxanthophyll crystals in plant oleoresins.
 8. The method of claim 1,wherein the scope of reaction time is 0.5-6.0 hours and the scope oftemperature is 60-90° C. when it has a isomerization reaction.
 9. Themethod of claim 1, wherein the described alcohol solvents are methanol,ethanol, isopropyl alcohol or propanol.
 10. The method of claim 2,wherein the adding volume of food grade alcohol solvents is 0.5-2.0times the volume of the same weight of plant oleoresins in step a). 11.The method of claim 10, wherein the used strong alkalies are NaOH, KOH,sodium methoxide or sodium ethoxide, the needed volume of alkalies is0.5-6.0 times the xanthophyll diester-containing plant oleoresins whilecarrying out soap-dissolution, and the used volume of alkalies is0.1-1.0 times the weight of the xanthophyll diester-containing plantoleoresins when it has an isomerization reaction.